Granulomas in Common Variable Immunodeficiency Display Different Histopathological Features Compared to Other Granulomatous Diseases

Abstract

Granulomatous disease affects up to 20% of patients with Common Variable Immunodeficiency (CVID). Granulomas are comprised of highly activated immune cells, and emerge in response to antigenic triggers. In CVID granulomas however, the underlying pathophysiology is unclear and the specific trigger remains unknown. Granuloma formation in CVID is often compared to sarcoidosis, although clinical context and prognosis differ, suggesting a different pathogenesis. The aim of this study was to investigate if the cellular organization and proteomics of granulomas in CVID is different from other granulomatous diseases. Therefore, tissue slides from formaldehyde fixed paraffin embedded biopsies obtained from patients with CVID, sarcoidosis, tuberculosis and foreign-material induced pseudo-sarcoidosis were stained with hematoxylin and eosin and assessed for histopathological characteristics. Targeted spatial protein analysis was performed, and immune fluorescent multiplex assays were used to analyze the cellular organization. Histological analysis revealed that CVID granulomas were smaller, less circumscribed, with fewer multinucleated giant cells and minimal fibrosis compared to the other granulomatous diseases. Spatial protein analysis showed that granulomas in all diseases expressed CD68, CD11c, CD44, CD127, and PD-L1. However in CVID, reduced expression of the fibrosis-related protein fibronectin, but enrichment of CD163, CD3 and FAPα inside CVID granulomas was observed. Immunofluorescence analysis conformed a different cellular organization in CVID granulomas with increased influx of neutrophils, macrophages, T and B lymphocytes. In conclusion, granulomas in CVID display a different histological and cellular organization with increased influx of myeloid and lymphoid cells, compared to sarcoidosis, tuberculosis and pseudo-sarcoidosis, indicating a distinct pathogenesis underlying granuloma formation.

Keywords: Common variable immunodeficiency; Granuloma; Histology; Sarcoidosis; Spatial protein expression.

Fig. 1

Representative HE stains and granuloma diameter (A) 20x Lymph node HE stains of representative biopsies of CVID, sarcoidosis (Sarc), PS and TB. (B) Granuloma size per disease group based on the diameter of 30 randomly selected granulomas for each disease group. Median and interquartile ranges are indicated. Kruskal-Wallis with Dunn’s multiple comparison correction, *=P < 0.05, **=P < 0.01, ****=P < 0.0001. (C) Discriminative capacity granuloma sizes to discriminate CVID granulomas from sarcoidosis, PS or TB, shown by ROC analysis. See also Table 2

Fig. 2

Spatial protein analysis of inter disease comparison granuloma versus surrounding. (A) Relative protein expression of 72 granulomas (3 granulomas per each of 6 patients per disease group) and the adjacent surrounding. Fold induction threshold was set to 1 (red line), significance threshold at 1.2 (green line) showing uncorrected P-values. Only significantly or borderline significant targets are indicated. (B) Absolute protein counts with median of significant protein expression commonalities in the granulomas of the four diseases. (C) Absolute protein counts with median of protein targets enriched in the granulomas versus the surrounding only in a subset of the volcano plots. (D) Absolute protein counts with median of protein targets enriched in the surrounding versus the granulomas only in a subset of the volcano plots. Statistical differences only indicated for expression in granuloma versus the surrounding per disease, with statistical testing by Mann-Whitney test with * = P ≤ 0.05, ** = P ≤ 0.01, *** = P ≤ 0.001

Fig. 3

Spatial protein analysis in the granulomas of CVID versus granulomas of sarcoidosis, PS and TB. (A) Relative protein expression of 72 granulomas, comparing CVID versus sarcoidosis, or PS or TB, with fold induction threshold set to 1 (red line), significance threshold at 1.2 (green line), showing uncorrected P-values. Only significantly altered or borderline significant targets are indicated. B-D) Absolute protein counts with median of significantly enriched protein differences between CVID versus either (B) sarcoidosis, (C) PS or (D) TB. Statistical testing was performed by Kruskall-Walis with Dunn’s multiple comparison correction, * = P ≤ 0.05, ** = P ≤ 0.01, *** = P ≤ 0.001

Fig. 4

Spatial protein analysis of surrounding areas of granulomas of CVID versus sarcoidosis, PS and TB. (A) Relative protein expression of 72 granulomas, comparing CVID versus sarcoidosis, or PS or TB, with fold induction threshold set to 1 (red line), significance threshold at 1.2 (green line), showing uncorrected P-values. Only significantly altered or borderline significant targets are indicated. (B) Absolute protein counts with median of significantly depleted protein differences in the surrounding areas of CVID versus sarcoidosis, PS or TB. Statistical testing was performed by Kruskall-Walis with Dunn’s multiple comparison correction, * = P ≤ 0.05, ** = P ≤ 0.01, *** = P ≤ 0.001

Fig. 5

Cellular distribution and quantification of myeloid, fibroblast-like and lymphoid cells in the granulomas and their surroundings. (A) Heat map showing the median of positive cell counts of the cellular markers per disease group (CVID, sarcoidosis, PS or TB), normalized towards the total cells detected in that region (granuloma or surrounding). B-D) 10x representative immunofluorescence single stains together with DAPI per IF multiplex assay performed, always showing the same biopsy sample per disease of all three IF assays, complemented with the quantified positive cell counts per total cells detected of the indicated regions and markers per disease group, with violin plots showing median and interquartile range; (B) DAPI = dark blue, CD68 = aqua, CD11c = yellow, PD-L1 = white, FAPα = red, SMA = green; (C) DAPI = dark blue, CD68 = aqua, CD163 = red, pERK1/2 = white, MPO = yellow, FAPa = green; (D) DAPI = dark blue, CD3 = red, CD4 = yellow, FOXP3 = aqua, CD20 = green, PD-1 = white. Statistical analysis with 2way anova, with Tukey’s multiple comparisons test, * = P ≤ 0.05, ** = P ≤ 0.01, *** = P ≤ 0.001

Fig. 6

Summary of immune histologic hallmarks of CVID, sarcoidosis, pseudo sarcoidosis (PS) and tuberculosis (TB) granulomas. The graphical overview shows the investigated hallmarks that differed distinctively in CVID. The observed presence of these hallmarks in CVID, sarcoidosis, PS or TB granulomas are indicated with the blue gradient. Created with www.Biorender.com